HEAT-SHOCK-PROTEIN-80 OF NEUROSPORA-CRASSA, A CYTOSOLIC MOLECULAR CHAPERONE OF THE EUKARYOTIC STRESS-90 FAMILY, INTERACTS DIRECTLY WITH HEAT-SHOCK-PROTEIN-70

The subunit structure of Hsp80, the most abundant heat-shock protein o f Neurospora crassa, was examined by chemical cross-linking of the pur ified protein in vitro. Resolution of glutaraldehyde-treated Hsp80 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis SDS-PAGE sug gests that the native state of this protein is a tetramer; the relativ e proportion of cross-linked species, estimated by the fraction of pro tein recovered in each category, is consistent with a dimer-of-dimer s tructure. Upon interaction with nucleotides, higher order cross-linked oligomers were detected, indicating ligand-induced conformational cha nges. The effect of nucleotides was also monitored by following trypto phan fluorescence: CTP, UTP, and NAD led to fluorescence quenching, th e effect of CTP being the most pronounced. As individual molecular cha perones often act in concert with cochaperones, interaction between th e two major cytosolic stress proteins-Hsp80 and Hsp70-was examined. Pu rified Hsp70 was immobilized on ATP-agarose and purified Hsp80 was app lied to the Hsp70-saturated matrix; while Hsp80 did not bind to ATP-ag arose by itself, it was bound strongly by immobilized Hsp70. The [Hsp7 0-Hsp80] complex was eluted with ATP and coelution of both proteins wa s confirmed by Western blot analysis, using specific polyclonal antibo dies raised against each protein. The physical association of stress-i nducible Hsp70 and Hsp80 was verified by interprotein cross-linking in vitro followed by immunoblot analysis and by immunoprecipitation.